Stationary phase characteristics

Metal chelate chromatography was originally developed using low pressure stationary phases such as agarose (Hemdan and Porath, 

A list of compounds separated on Enatiopac (silica-glycoprotein) (reproduced from LKB technical brochure, with permission) 

The methods which are used for the preparation of HPLEC stationary phases are similar to those which have been developed for soft gel matrices by treating controlled pore glass with 3-(2-aminoethy-lamino)propyl-trimethoxysilane and subsequently perfusing the column with copper sulphate, a copper-chelate support is prepared (Masters and Leyden, 1978). Direct treatment of silica with copper sulphate can also be used (Caude and Foucault, 1979) (Fig. 9.6). Methods used in the preparation of these stationary phases can be found in the literature (Sugden et al., 1980 Caude et al., 1984). 

The use of mobile phase chiral additives for the separation of amino acids and dansyl amino acids has been reviewed previously (Gil-Av 

D-lysine 2, L-lysine 3, D-arginine 4, glycine 5, D-alanine, D-serine 6, D-threonine 7, D-arginine 8, L-threonine, L-alanine, D-hi tidine 9, D-cysteic acid 10, L-cysteic acid  

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CEC is often inappropriately presented as a hybrid method that combines the capillary column format and electroosmotic flow employed in high-performance capillary electrophoresis with the use of a solid stationary phase and a separation mechanism, based on specific interactions of solutes with the stationary phase, characteristic of HPLC. Therefore CEC is most commonly implemented by means typical of both HPLC (packed columns) and CE (use of electrophoretic instrumentation). To date, both columns and instrumentation developed specifically for CEC remain scarce. 

The speed of a chromatographic separation is fixed by the particle size, the stationary phase characteristics, the available pressure, the solvent viscosity, the solute diffusivity, the a values of the critical pair, and extracolumn dispersion. One way to achieve faster separations is to reduce the particle size of the stationary phase. However, if material of smaller diameter is packed into a conventional size column, the backpressure will become prohibitively high. Thus, in a compromise between speed and optimum performance, narrow (<2 mm) columns packed with small 3-5 ju.m diameter particles have been developed. 

The first chiral separation using pSFC was published by Caude and co-workers in 1985 [3]. pSFC resembles HPLC. Selectivity in a chromatographic system stems from different interactions of the components of a mixture with the mobile phase and the stationary phase. Characteristics and choice of the stationary phase are described in the method development section. In pSFC, the composition of the mobile phase, especially for chiral separations, is almost always more important than its density for controlling retention and selectivity. Chiral separations are often carried out at T < T-using liquid-modified carbon dioxide. However, a high linear velocity and a low pressure drop typically associated with supercritical fluids are retained with near-critical liquids. Adjusting pressure and temperature can control the density of the subcritical/supercritical mobile phase. Binary or ternary mobile phases are commonly used. Modifiers, such as alcohols, and additives, such as adds and bases, extend the polarity range available to the practitioner. 

Reasons for IGC s higher profile in the technical literature include convenience and economics of operation. The basic tools for IGC are inexpensive, rugged, widely available, and as well suited for routine laboratory applications, as they are for demanding fundamental research. IGC data may be collected quite rapidly over extended temperature ranges. A variety of probes may be used in the mobile phase to elucidate the characteristics of the stationary phase, characteristics that otherwise are only obtained at far greater expenditure of time and money. 

In GC capacity factors, retention and selectivity are controlled by adjusting the column temperature and stationary phase characteristics. In LC change in the composition of the eluant serves both purposes more effectively and thus solvent programming (otherwise known as gradient elution) is used in HPLC where temperature programming would be employed in GC. 

E. Pefferkorn, Ed., Interfacial Phenomena in Chromatography, Marcel Dekker Inc, New York, 1999. pp. 1-171, Part 1 Stationary Phase Characteristics. 

In the approach of Rohrschneider, the difference in the retention index value for polar phase (A/) is represented as a series of terms composed of solute-specific contributions (a,..., c) and the stationary phase characteristic term (x, .. which can be written as " ... 

As already mentioned, the number of phases in CGC can be reduced further due to the high efficiency those columns offer. Si ark et al. [211 studied stationary-phase characteristics in depth and concluded that the order of optimized phases for CGC, all higher molecular mass crosslinkable gums, is... 

Another important characteristic of a gas chromatographic column is the thickness of the stationary phase. As shown in equation 12.25, separation efficiency improves with thinner films. The most common film thickness is 0.25 pm. Thicker films are used for highly volatile solutes, such as gases, because they have a greater capacity for retaining such solutes. Thinner films are used when separating solutes of low volatility, such as steroids. 

Hydrophobic Interaction Chromatography. Hydrophobic interactions of solutes with a stationary phase result in thek adsorption on neutral or mildly hydrophobic stationary phases. The solutes are adsorbed at a high salt concentration, and then desorbed in order of increasing surface hydrophobicity, in a decreasing kosmotrope gradient. This characteristic follows the order of the lyotropic series for the anions ... 

The theoretical treatment given above assumes that the presence of a relatively low concentration of solute in the mobile phase does not influence the retentive characteristics of the stationary phase. That is, the presence of a small concentration of solute does not influence either the nature or the magnitude of the solute/phase interactions that determine the extent of retention. The concentration of solute in the eluted peak does not fall to zero until the sample volume is in excess of 100 plate volumes and, at this sample volume, the peak width has become about five times the standard deviation of the normally loaded peak. 

Figure 8.17 Schematic diagram of a cross-section (a) tlvough the clamped plates, and views from above (b-d) of coupled plates serially connected to achieve multidimensional separation with stationary phases with different characteristics (hatched lines, glass plate light shading, stationary phase A dark shading, stationary phase B wavy lines, stationary phase C).

Selectivity The characteristics of the stationary phase that determine how far apart the peak maxima of two components will be separated. 

The vast majority of modem liquid chromatography systems involve the use of silica gel or a derivative of silica gel, such as a bonded phase, as a stationary phase. Thus, it would appear that most LC separations are carried out by liquid-solid chromatography. Owing to the adsorption of solvent on the surface of both silica and bonded phases, however, the physical chemical characteristics of the separation are more akin to a liquid-liquid distribution system than that of a liquid-solid system. As a consequence, although most modern stationary phases are in fact solids, solute distribution is usually treated theoretically as a liquid-liquid system. 

Returning now to the subject of the chapter, in addition to appropriate retentive characteristics, a potential stationary phase must have other key physical characteristics before it can be considered suitable for use in LC. It is extremely important that the stationary phase is completely insoluble (or virtually so) in all solvents that are likely to be used as a mobile phase. Furthermore, it must be insensitive to changes in pH and be capable of assuming the range of interactive characteristics that are necessary for the retention of all types of solutes. In addition, the material must be available as solid particles a few microns in diameter, so that it can be packed into a column and at the same time be mechanically strong enough to sustain bed pressures of 6,000 p.s.i. or more. It is clear that the need for versatile interactive characteristics, virtually universal solvent insolubility together with other critical physical characteristics severely restricts the choice of materials suitable for LC stationary phases. 

Qualitative (identification) applications depend upon the comparison of the retention characteristics of the unknown with those of reference materials. In the case of gas chromatography, this characteristic is known as the retention index and, although collections of data on popular stationary phases exist, it is unlikely that any compound has a unique retention index and unequivocal identification can be effected. In liquid chromatography, the situation is more complex because there is a much larger number of combinations of stationary and mobile phases in use, and large collections of retention characteristics on any single system do not exist. In addition, HPLC is a less efficient separation... 

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